Project Summary Our overall hypothesis is that the local killing of mammary epithelial cells will be as effective as prophylactic mastectomy in preventing breast cancer, but with minimal side effects. Our long-term goal is to develop a minimally invasive intraductal injection procedure that fills the entire ductal tree with a solution to locally kill the mammary epithelial cells and thereby prevent tumor formation before cells become malignant. Our objectives for this preclinical study are a) to test this concept in a Brca1-deficient mouse model of breast cancer using a chemical ablation approach with ethanol (EtOH) as the cell-killing compound and b) to begin to assess the scalability of this procedure by using rats as an intermediate model. Our preliminary data show the feasibility of filling the entire ductal tree with a solution of up to 70% ethanol and that such a solution can locally kill mammary epithelial cells. However, we have not tested the cell-killing ability of other ethanol concentrations nor have we studied long-term effects of this procedure. For mouse studies, we will first establish the optimal ethanol concentration that achieves maximal mammary epithelial cell death while minimizing collateral tissue damage. To this end, we will collect mammary gland tissue samples at time points from 1 day to two months after intraductal injection, and we will determine the effects of ethanol concentrations from 50% to 70% via histological and immunohistochemical analyses. Then, we will use the MMTV-Cre;Brca1fl/fl;p53+/- breast cancer model, which is relevant to high-risk individuals with mutated BRCA genes. Up to 8 mammary glands in each female animal (n = 20 per group) will be injected with the optimal ethanol concentration or phosphate-buffered saline (control). We expect that tumors will not form in mammary glands injected with the ethanol solution. We will use the log-rank test and logistic regression with random effect adjustment to analyze the difference in tumor latency and tumor incidence, respectively, between uninjected and injected mammary glands within each group and between groups. For rat studies, Sprague-Dawley rats will be injected with a size-proportional volume of the optimized ethanol solution to determine whether the procedure has the same effects in rats as in mice. We will use the same experimental design and tissue analyses as described for mice. Positive results from this preclinical study will provide a strong proof-of-concept toward translating this intraductal procedure to humans. This procedure could lead to a breakthrough in breast cancer prevention by providing a universal prophylactic intervention, one not just for high-risk individuals; by improving an individual's quality of cancer-free life; and by decreasing the personal and societal costs of breast cancer. Given the existing clinical uses of ethanol, ethanol- based ablation protocols could be readily implemented in clinical trials for breast cancer prevention. This study could also stimulate the evaluation of other chemical and/or thermal ablation strategies. Intraductal delivery of a cell-killing solution could be achieved by repurposing or modifying FDA-approved devices for ductal lavage or ductography.